DESCRIPTION (taken from the application): Smooth muscle cells are found in the contractile apparatus of many tissues and organs, including the arteries, bronchi, and uterus. In smooth and non-muscle cells contraction is primarily regulated through phosphorylation by the myosin light chain kinase and dephosphorylation by the myosin phosphatase of Serl9 of the myosin regulatory light chain. A second mechanism is thought to involve caldesmon, a protein found in association with the actin thin filaments. In this project we take a two-pronged crystallographic approach to investigate the structural basis of smooth muscle regulation. Based on the crystal structure of monomeric ADP-actin that we have determined recently, we now propose to extend this investigation to study the structure-function relationship of different actin isoforms and the role of different nucleotide states on the structure of actin. This will help understand the role of ATP hydrolysis in controlling the dynamics of actin filaments and characterize, for the first time, the atomic structure of a smooth actin isoform. We have been successfully using this actin as a sort of a "crystallization carrier" to bring the actin-binding domain of caldesmon to co-crystallize. This approach is aimed at overcoming difficulties encountered with the crystallization of flexible molecules such as caldesmon. The underlying hypothesis is that the structure of the caldesmon fragment will be stabilized by its interactions with actin within the complex. Co-crystals are available, but they require further improvement. Our third aim, which can be viewed as the sum of four sub-aims, deals with the study of the trimeric myosin phosphatase. Difficulties encountered with the expression of the various phosphatase subunits have so far prevented large-scale crystallization trials. We have been able to overcome this problem by (co-)expressing in E. coli a 36 and 50kDa N-terminal fragments of the phosphatase targeting subunit alone and in conjunction with the catalytic subunit. Although crystals are not yet available for this aim, we have tried to dilute the risks associated with crystallization by working in parallel on four major structures, namely we propose to crystallize the 36 and 50kDa fragments of the targeting subunit alone as well as their respective complexes with the catalytic subunit. The work in this project will be carried out in close collaboration with other projects of this program project.